Approximately 50% of all mammalian peptide hormones possess a C-terminal amino acid amide. The C-terminal amide is produced in vivo by the post-translational, oxidative cleavage of an inactive glycine-extended prohormone to yield the active, alpha-amidated peptide and glyoxylate. The enzyme catalyzing this reaction is peptidylglycine alpha-amidating monooxygenase (PAM). Since the discovery of PAM in the early 1980's, a considerable body of mechanistic and structure/function data has been generated in an attempt to better define the details of the PAM reaction. Despite these efforts, there are still crucial unanswered questions regarding the number of ascorbate-binding sites, the rates of electron transfer, and the potential formation of a substrate based radical that preclude a comprehensive understanding of the PAM reaction. The overall goal of this project is probe the fundamental mechanism of PAM catalysis using an array of modern biochemical and spectroscopic techniques. Specifically, the aims of this proposal are to: 1. Determine the number of ascorbate binding sites by equilibrium binding using radiolabeled mimosine, a PAM inhibitor competitive vs. ascorbate. 2. Measure the substituent-dependence of the inactivation rates of PAM by a series ring-substituted 4-phenyl-3-butenoates to establish the electronic nature of the transition state for inactivation. 3. Rapid freeze-quench ERR studies to (a) measure the rates of PAM-bound Cu(ll) reduction and Cu(l) reoxidation and to (b) identify and characterize ERR active intermediates that form during the catalytic cycle. PAM is copper-dependent and is, thus, a member of the copper monooxygenase family. Like PAM, all the copper monooxygenase catalyze a reaction of considerable biological significance and utilize copper for the reductive activation of O2 linked to the insertion of an oxygen atom into a C-H bond. The molecular details of this chemistry are incompletely understood. Our work on PAM will contribute valuable new mechanistic information regarding the copper monooxygenases that will provide crucial guidance for the future development of drugs potentially useful for the treatment of diseases linked to this family of enzymes.